Nicholas Hoe

Transcriptional profiling of MnSOD-mediated lifespan extension in Drosophila reveals a species-general network of aging and metabolic genes

BackgroundSeveral interventions increase lifespan in model organisms, including reduced insulin/insulin-like growth factor-like signaling (IIS), FOXO transcription factor activation, dietary restriction, and superoxide dismutase (SOD) over-expression. One question is whether these manipulations function through different mechanisms, or whether they intersect on common processes affecting aging.ResultsA doxycycline-regulated system was used to over-express manganese-SOD (MnSOD) in adult Drosophila, yielding increases in mean and maximal lifespan of 20%. Increased lifespan resulted from lowered initial mortality rate and required MnSOD over-expression in the adult. Transcriptional profiling indicated that the expression of specific genes was altered by MnSOD in a manner opposite to their pattern during normal aging, revealing a set of candidate biomarkers of aging enriched for carbohydrate metabolism and electron transport genes and suggesting a true delay in physiological aging, rather than a novel phenotype. Strikingly, cross-dataset comparisons indicated that the pattern of gene expression caused by MnSOD was similar to that observed in long-lived Caenorhabditis elegans insulin-like signaling mutants and to the xenobiotic stress response, thus exposing potential conserved longevity promoting genes and implicating detoxification in Drosophila longevity.ConclusionThe data suggest that MnSOD up-regulation and a retrograde signal of reactive oxygen species from the mitochondria normally function as an intermediate step in the extension of lifespan caused by reduced insulin-like signaling in various species. The results implicate a species-conserved net of coordinated genes that affect the rate of senescence by modulating energetic efficiency, purine biosynthesis, apoptotic pathways, endocrine signals, and the detoxification and excretion of metabolites.

Alteration of Drosophila life span using conditional, tissue-specific expression of transgenes triggered by doxycyline or RU486/Mifepristone.

The conditional systems Tet-on and Geneswitch were compared and optimized for the tissue-specific expression of transgenes and manipulation of life span in adult Drosophila. Two versions of Tet-on system reverse-tetracycline-Trans-Activator (rtTA) were compared: the original rtTA, and rtTAM2-alt containing mutations designed to optimize regulation and expression. The rtTAM2-alt version gave less leaky expression of target constructs in the absence of doxycyline, however the absolute level of expression that could be achieved was less than that produced by rtTA, in contrast to a previous report. Existing UAS-rtTAM2-alt insertions were re-balanced, and combined with several tissue-general and tissue-specific GAL4 driver lines to yield tissue-specific, doxycyline-inducible transgene expression over three orders of magnitude. The Geneswitch (GS) system also had low background, but the absolute level of expression was low relative to Tet-on. Consequently, actin5C-GS multi-insert chromosomes were generated and higher-level expression was achieved without increased background. Moderate level over-expression of MnSOD has beneficial effects on life span. Here high-level over-expression of MnSOD was found to have toxic effects. In contrast, motor-neuron-specific over-expression of MnSOD had no detectable effect on life span. The results suggest that motor-neuron tissue is not the essential tissue for either MnSOD induced longevity or toxicity in adult males.

A novel proteomics-based clinical diagnostics technology identifies heterogeneity in activated signaling pathways in gastric cancers.

Purpose The aim of this study was to utilize the proteomics-based Collaborative Enzyme Enhanced Reactive (CEER) immunoassay to investigate protein tyrosine phosphorylations as diagnostic markers in gastric cancers (GCs). Experimental Design Protein lysates from fresh-frozen 434 advanced stage GCs were analyzed for phosphorylation of HER1, HER2, p95HER2, HER3, cMET, IGF1R and PI3K. The pathway activation patterns were segregated based on the tumor HER2 status. Hierarchical clustering was utilized to determine pathway coactivations in GCs. Prognostic value of pathway activation patterns was determined by correlating disease-free survival times of the various GC subgroups using Kaplan-Meier survival analysis. CEER was also used to determine the presence of tyrosine phosphorylated signaling cascades in circulating tumor cells (CTCs) and ascites tumor cells (ATCs). Results Utilizing a novel diagnostics immunoassay, CEER, we demonstrate the presence of p95HER2 and concomitantly activated signaling pathways in GC tumor tissues, CTCs and ATCs isolated from GC patients for the first time. p95HER2 is expressed in ∼77% of HER2(+) GCs. Approximately 54% of GCs have an activated HER1, HER2, HER3, cMET or IGF1R and demonstrate a poorer prognosis than those where these receptor tyrosine kinases (RTKs) are not activated. Hierarchical clustering of RTKs reveals co-clustering of phosphorylated HER1:cMET, HER2:HER3 and IGF1R-PI3K. Coactivation of HER1 with cMET renders GCs with a shorter disease-free survival as compared to only cMET activated GCs. Conclusions Our study highlights the utility of a novel companion diagnostics technology, CEER that has strong implications for drug development and therapeutic monitoring. CEER is used to provide an increased understanding of activated signaling pathways in advanced GCs that can significantly improve their clinical management through accurate patient selection for targeted therapeutics.

Hydrogen Peroxide Stimulates Activity and Alters Behavior in Drosophila melanogaster

Circadian rhythms in animals are regulated at the level of individual cells and by systemic signaling to coordinate the activities of multiple tissues. The circadian pacemakers have several physiological outputs, including daily locomotor rhythms. Several redox-active compounds have been found to function in regulation of circadian rhythms in cells, however, how particular compounds might be involved in regulating specific animal behaviors remains largely unknown. Here the effects of hydrogen peroxide on Drosophila movement were analyzed using a recently developed three-dimensional real-time multiple fly tracking assay. Both hydrogen peroxide feeding and direct injection of hydrogen peroxide caused increased adult fly locomotor activity. Continuous treatment with hydrogen peroxide also suppressed daily locomotor rhythms. Conditional over-expression of the hydrogen peroxide-producing enzyme superoxide dismutase (SOD) also increased fly activity and altered the patterns of locomotor activity across days and weeks. The real-time fly tracking system allowed for detailed analysis of the effects of these manipulations on behavior. For example, both hydrogen peroxide feeding and SOD over-expression increased all fly motion parameters, however, hydrogen peroxide feeding caused relatively more erratic movement, whereas SOD over-expression produced relatively faster-moving flies. Taken together, the data demonstrate that hydrogen peroxide has dramatic effects on fly movement and daily locomotor rhythms, and implicate hydrogen peroxide in the normal control of these processes.

Abstract 1469: Integrated analysis of microRNA, mRNA, and protein expression utilizing MultiOmyxTMand NanoStringTMfrom formalin-fixed paraffin-embedded, lung, head and neck, breast, and melanoma tumors

Cancer is characterized as a loss of normal cellular regulation, due to accumulation of mutations and disruption of complex biological pathways. MicroRNAs (miRNAs) regulation of co-stimulatory and immune checkpoint pathways have been implicated as one of the potential mechanisms for cancer evasion in immuno-oncology. It is estimated that 30% of all mRNA expression may be regulated by miRNAs, and some are either oncogenic or tumor suppressive. Complexity of miRNA regulation highlights the need for integrated assays, providing direct correlation between miRNA and mRNA, and protein expression. From a single 4 µm FFPE section, MultiOmyxTM hyperplexed immunofluorescent assay (demonstrated to stain up to 60 protein biomarkers) is utilized to measure CD3, CD4, CD8, CD16, CD56, Granzyme B, FoxP3, ICOS, OX40, OX40L, PD1, PDL1, HLA-DR, and Ki67 protein expression. From an adjacent 10 µm section, NanoStringTM nCounter PanCancer Immune Profiling Panel and Human v3 miRNA expression panel were utilized to comprehensively profile the expression of 770 mRNA and 800 miRNA. Integrating MultiOmyx and NanoString technologies, the current study measured miRNA, mRNA, and protein expression in lung, head and neck, breast, and melanoma samples. For each indication, three samples were selected from a larger sample set, based on high protein expression of lymphocytes and macrophage markers (CD3, CD4, CD8, CD56, CD16), co-stimulator markers (ICOS, OX40), and immune checkpoint markers (PD1, PD-L1). Protein expression results indicate positive correlation between expression of ICOS and OX40 with higher infiltration of Thelper (CD3+CD4+), Tcytotoxic (CD3+CD8+), and effector T cells (CD3+CD8+Granzyme B). NanoString normalized mRNA counts for the protein biomarkers profiled indicate that all markers except for HLA-DR belong to low expressers group with counts ranging from 20-700. Comparison of protein expression to mRNA counts revealed inconsistencies in modulated markers (PD1, PD-L1, ICOS, OX40) which are attributed to differences in population of cells between the two sections. Direct assessment of up regulation of miRNA and down regulation of target mRNA could not be made for miRNAs reported in literature as negative regulator of PD-L1 (miR-34a, 34b, 34c), PD1 (miR-28, miR-107), FoxP3 (miR-210, miR-24, miR-31), and ICOS (miR-101). Analysis of multiple miRNAs (combinatorial targeting) mapped in context to mRNA, and their respective protein expression will be presented from lung, head and neck, breast, and melanoma cancer samples. Citation Format: Qingyan Au, RaghavKrishna Padmanabhan, Nam Tran, Lakshmi Chandramohan, Nicholas Hoe. Integrated analysis of microRNA, mRNA, and protein expression utilizing MultiOmyxTM and NanoStringTM from formalin-fixed paraffin-embedded, lung, head and neck, breast, and melanoma tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1469. doi:10.1158/1538-7445.AM2017-1469

Abstract 5135: Detection of IFNγ induced PDL1 expression by combinedin situRNA analysis and protein profiling from a single FFPE slide

PD1 ligands (PDL1) are often upregulated on the cell surface of many different tumors. The primary role of PDL1 in cancer is to inhibit T-cell mediated immune response. Two general mechanisms for PDL1 expression on tumor cells have been proposed. Innate immune resistance, in which PDL1 expression is induced by the constitutive oncogenic signaling, and adaptive immune resistance, in which PDL1 expression is induced by T-cells releasing interferon-γ (IFNγ) and activating the STAT signaling pathway. In order to differentiate between these two mechanisms, IFNγ mRNA expression is measured as an effective alternative to detecting IFNγ protein. Detection of cytokines by IHC is challenging as secreted proteins are widely diffused and the associated staining pattern appears to lack cellular specificity. RNAscope RNA in situ hybridization (ISH) assay is utilized to measure Interferon-γ (IFNγ) mRNA expression, and MultiOmyxTM multiplexed assay (demonstrated to stain up to 60 protein biomarkers) is utilized to measure CD3, CD4, CD8, CD56, CD68, PD1, and PDL1 protein expression. In this study, combined MO and RNAscope ISH assays, enabled identification of individual cells with characteristic mRNA and protein expression profile. The MultiOmyx assay utilizes a pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Each Cy-dye conjugated antibody recognizes different target proteins. Each round of staining is imaged and followed by novel dye inactivation chemistry, enabling repeated rounds of staining. RNAscope is a novel RNA ISH assay capable of single-molecule detection in individual cells, utilizing hybridization mediated signal amplification. The assay utilizes a pair of RNA target specific oligonucleotide probes, which sequentially hybridize to preamplifier, amplifier, and fluorophore label probes. Utilizing MultiOmyx and RNAscope assays, this study proposed to profile both RNA and protein expression in lung, breast, melanoma, colorectal, esophageal, and prostate cancer samples. Differentiating PDL1 expression induced in response to inflammatory signals produced by an activated T-cell, from PDL1 expression induced by constitutive oncogenic signaling, has potential implications in effectiveness of PD1 blockade therapy. According to the proposed mechanisms, PD1 blockade as a mono therapy may only benefit individuals with strong endogenous immune response. In individuals with weak endogenous immune response, combinational therapies consisting of both immune activation and PD1-pathway blockade may be more effective than either mono therapy alone. Citation Format: Qingyan Au, Kathy Nguyen, Michael S. Lazare, Edward J. Moler, Nicholas Hoe. Detection of IFNγ induced PDL1 expression by combined in situ RNA analysis and protein profiling from a single FFPE slide. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5135.

Abstract 4640: Highly sensitive proximity mediated immunoassay can determine functional VEGFR2 status in clinical samples for routine clinical uses.

VEGFR2 is the primary signaling receptor for VEGF-A, and is considered to be the major initiator of angiogenesis. Binding of the growth factor activates VEGFR2 via tyrosine phosphorylation and results in induction of angiogenic and lymphangiogenic signals. An understanding of the VEGFR2 status of human tumors may help to identify patients who would benefit most from treatment with VEGFR2 inhibitors. Currently there are several drugs targeting VEGFR2 either approved for clinical use or in various stages of clinical trials. Bevacizumab, a monoclonal antibody to VEGF-A is used to treat patients with lung, colon and breast cancer. Sunitinib targeting VEGFR2, PDGFRβ and c-Kit was approved for treating carcinoid tumors in the gastro-intestinal stroma. VEGFR2 profiling could be used to monitor the efficacy of anti-angiogenic treatment in cancer patients. Here we report the development of immunoarray assays for functional characterization of VEGFR2 and their application in profiling VEGFR2 status in preclinical and clinical specimens. The CEERTM (Collaborative Enzyme Enhanced Reactive-immunoassay) assays use an antibody-microarray based platform which utilizes the formation of a unique “triple-antibody-enzyme-channeling” immuno-complex capable of measuring expression (total) and activation (phospho) of VEGFR2 in tumor tissues or surrogate tissues with limited availability. CEERTM platform can detect levels of expression and activation of VEGFR2 in as little as 10 cells due to its unique proximity channeling assay configuration. Specimens are analyzed along with standards so quantitative read out can be compared longitudinally. Assay specificity was established by comparing levels of phosphorylated VEGFR2, between the non-treated and treated with VEGF. Phosphorylation levels were increased in VEGFR2 positive cell lines HUVEC and H441 when treated, while VEGFR2 negative cell lines MDA-MB-134 IV, BT474, SNU16, Ramos lymphoma, and K562 did not. In clinical setting, robust quantifiable signals were detected in FNAs or core biopsy tissues obtained from various cancer patients. Ongoing analysis of clinical samples from colorectal, lung and renal cancer patients indicates an increase of VEGFR2 phosphorylation in tumor samples compared to healthy tissues and a reduction of VEGFR2 phosphorylation post VEGFR2-targeted therapy in some patients. 47% (18/38) of NSCLC patients have shown evidence of activated VEGFR2, and it would be interesting to determine if patients with activated VEGFR2 respond to Bevacizumab. The CEER-based VEGFR2 assay can provide quantitative analysis of clinical samples with limited availability providing critical information for developing effective strategies for treatment selection, monitoring drug response and overcoming potential resistance in cancer patients. Citation Format: Kelly D. Hester, Nicholas Hoe, Jinyao Zhou, Crystal Kuy, Liching Zhang, Phillip Kim, Xinjun Liu, Sharat Singh. Highly sensitive proximity mediated immunoassay can determine functional VEGFR2 status in clinical samples for routine clinical uses. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4640. doi:10.1158/1538-7445.AM2013-4640

Abstract 4273: FGFR inhibition modulates ErbB receptor tyrosine phosphorylation in FGFR amplified cell lines.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases (RTKs) are currently under investigation as therapeutic targets for the treatment of breast cancer. FGFR1 amplifications range from 7.5-17% in breast cancers and are associated with a shorter overall survival. FGFR1 amplified cell lines drive activation of AKT and ERK pathways and are highly sensitive to FGFR1 inhibition, suggesting an oncogenic addiction to FGFR1. FGFR2 has been reported to be activated in lapatinib-resistant, HER2-positive cells. Likewise, FGFR3 expression levels are often elevated in tamoxifen-resistant, ER positive patients. FGFR 4 overexpression correlated with poor response to chemotherapy due to activation of MAPK and increase in B-cell lymphoma extra large (BCL-XL) levels. Using a highly sensitive, novel proximity mediated immuno-microarray, Collaborative Enzyme Enhanced Reactive-immunoassay (CEERTM), this study seeks to determine whether FGFR signal transduction pathway inhibition modulate ErbB receptor tyrosine kinases and AKT/ERK signal transduction pathway in cell lines expressing varying levels of FGFR and ErbB receptors. FGFR 1, 2, 3, and 4 amplified cell lines (MDA-MB-134VI (KRAS), SNU16, RT112, and MDA-MB-453) were individually treated with varying dosages (1, 10, 100, and 1000nM) of pan FGFR inhibitors (AZD4547 and Ponatinib-AP24534) in presence of corresponding FGF ligand (FGF1, FGF7, FGF9, and FGF19). Expression and activation of FGFR 1, 2, 3, 4 and EGFR, HER2, HER3, HER4 and downstream signaling proteins FRS2, AKT, ERK, MEK, and RSK were measured utilizing CEERTM. Both FGFR inhibitors sufficiently inhibited phosphorylation of FGFRs in each of the four FGFR amplified cell line. In MDA-MB-453, FGFR4 inhibition increased phosphorylation of HER2, and HER3 along with AKT. In MDA-MB-134, FGFR1 inhibition decreased levels of HER3 phosphorylation along with reduction in phosphorylated ERK, MEK, and RSK. In SNU16, FGFR2 inhibition led to a decrease in EGFR, HER2, and HER3 phosphorylation along with reduction in phosphorylated MEK, ERK, and RSK. Analysis of RT112 and additional cell lines with varying levels of ErbB and FGFR receptor tyrosine kinases are currently in progress. Our data suggests that FGFR inhibition may modulate parallel / compensatory ErbB and other signaling pathway as observed in this study. Utilizing CEERTM, comprehensive profiling may provide unique insight into potential feedback mechanisms in each patient and evidence for rational selection of appropriate combinational therapies. Citation Format: Nicholas Hoe, Kelly Jin, Yating Ma, Crystal Kuy, Michael Mateling, JinYao Zhou, Richard Kirkland, Saswati Hazara, Phillip Kim, Xinjun Liu, Sharat Singh. FGFR inhibition modulates ErbB receptor tyrosine phosphorylation in FGFR amplified cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4273. doi:10.1158/1538-7445.AM2013-4273